Liver transplantation is the only treating method which can be utilized for patients with liver-based metabolic diseases or hepatic insufficiency. However, the treatment has problems such as shortage of donor livers, considerable postoperative lethality accompanied with operative risk, high costs and use of immunosuppressant over a long period. Recently, isolated hepatocyte transplantation or a bio-artificial liver with living hepatocytes is desired as filler until liver transplantation or regeneration of liver. Advantages of the isolated hepatocyte transplantation or the bio-artificial liver include that it is economical compared with an operation of liver transplantation, that the risk is few, and the like. In the isolated hepatocyte transplantation or the bio-artificial liver, a clinical use thereof is also limited because of the shortage of donor livers.
As an alternative to the isolated hepatocyte, there includes a liver cell-line which can be proliferated in large numbers in vitro, which maintains a property of the isolated hepatocyte and further which can provide a metabolic supplement after transplantation. It is expected that an establishment of the liver cell-line enabling to proliferate in large numbers and having a high-level liver function, and a development of a bank thereof enable the transplantation of required amount of liver cell as need arises and dissolve the shortage of donor livers.
It is known that a cell-line which maintains appropriate function for differentiation can be produced by transferring oncogenes to immortalize cells (K. A. Westerman, et al., Proc. Natl. Acad. Sci., USA., vol 93, 8971, (1996)). However, in case where the immortalizated cell-line is infused into a living body or applied to a extracorporeal assist device such as the bio-artificial liver, there is possibility that a patient is exposed to unexpected risk of malignant transformation. It is not assured that the transplanted cell is finally rejected, even though a heterozoic cell or an incompatible homologous human cell is employed. In human, a stable chimera state with the heterozoic cell and accidental engraftment of HLA incompatible homologous tumor are reported (Gartner, et al., N. Eng. J. Med. Vol. 335, 1494, (1996); K. Paradis, et al., Science, vol. 285, 1236, (1999)). Therefore, it is desired that high-safety liver cells can be easily available in large scale.
In addition, in the aspect of other than purpose to treat liver-based diseases, needs for establishment of the normal liver cell-line enabling to proliferate in large numbers and the cell bank thereof is increased. Mainly, there include utilization for 1) an assay model for human drug metabolism, 2) a development of new drug, and 3) an infection model of human hepatitis.
However, in the conventional culture technique, it is difficult to proliferate the high-safety liver cells in large numbers.
In view of the above problem, the present invention purpose to provide a method for proliferating a mammalian liver cell, which enables the limitless proliferation. Moreover, the present invention purpose to provide a liver cell obtained by the said method, and use of the liver cell such as a treating agent and an artificial liver.